Download The importance of chromosomes from the sixth homeologic group in

J Appl Genetics (2013) 54:179–184
DOI 10.1007/s13353-013-0144-2
PLANT GENETICS • SHORT COMMUNICATION
The importance of chromosomes from the sixth homeologic
group in the restoration of male fertility in winter triticale
with Triticum timopheevii cytoplasm
Stefan Stojałowski & Aleksandra Bobrowska &
Monika Hanek & Beata Myśków
Received: 5 December 2012 / Revised: 27 February 2013 / Accepted: 1 March 2013 / Published online: 29 March 2013
# The Author(s) 2013. This article is published with open access at Springerlink.com
Abstract The sterilising cytoplasm from Triticum timopheevii
is presently considered to be the most promising as regards to
the seed production of triticale hybrid cultivars. This study was
aimed at the utilisation of Diversity Arrays Technology (DArT)
for the preliminary identification of genomic regions with loci
controlling male sterility/fertility in the field-grown F2 generation of the interline hybrid between male sterile line CMSSalvo 15/1 and restorer line Stan I. The fertility of plants was
examined by visual scoring as well as by the assessment of seed
setting within bagged spikes. For DNA analyses, 92 individuals representing opposite phenotypes (male sterile vs. fully
male fertile) were chosen from the whole F2 population, which
consisted of 414 plants. The constructed genetic map consists
of 759 DArT markers distributed in 24 linkage groups that
cover a distance of 974.4 cM. Application of the interval
mapping method and the Kruskal–Wallis test enabled the identification of six genomic regions engaged in the restoration of
male fertility within the mapping population. The most effective restorer genes were found on chromosomes of the sixth
homeologic group, i.e. on 6R (the most efficient), 6A and
6B. Additionally, linkage groups assigned to chromosomes
1BS, 3A and 3A/3B were important for the determination
of male fertility.
Keywords Cytoplasmic male sterility . Triticale . Triticum
timopheevii cytoplasm
Triticale (× Triticosecale Wittm.) is an artificial allo-hexaploid
species obtained by breeders as a result of intergeneric crosses
between wheat and rye. The first cultivars of this crop were
S. Stojałowski (*) : A. Bobrowska : M. Hanek : B. Myśków
Department of Plant Genetics, Breeding and Biotechnology,
West Pomeranian University of Technology, Słowackiego 17,
71-434 Szczecin, Poland
e-mail: [email protected]
registered over 50 years ago. Recently, this cereal has become
successively more common year on year. Over the last two
decades, breeders of triticale have become increasingly more
interested in the exploration of heterosis (Oettler et al. 2003,
2005; Góral et al. 2005; Tams et al. 2005). Practicable organisation of seed production for hybrid cultivars could be
achieved via application of the cytoplasmic male sterility
(CMS) system in triticale. Among the few sources of sterilising
cytoplasm available for this crop, the cytoplasm of Triticum
timopheevii Zhuk. is considered to be one of the most promising, due to its small number of deleterious effects (Cauderon
et al. 1985; Nalepa 1990; Spiss and Góral 1994). Utilisation of
the T. timopheevii CMS system in the breeding of hybrid
cultivars may be strongly facilitated by a better understanding
of the genetic mechanisms leading to microsporogenesis disorders in triticale plants carrying this cytoplasmic system.
Unfortunately, current knowledge about the genetic background of male sterility in this cereal is very limited. The
majority of research concerning genes restoring male fertility
in plants with T. timopheevii cytoplasm is focused on wheat.
The cytoplasm originating from Triticum timopheevii is also
important in wheat hybrid cultivar breeding programmes, as it
leads to male sterility symptoms in this cereal. Curtis and
Lukaszewski (1993) reported on the capability of a rye gene
located on the long arm of the 6R chromosome to restore male
fertility in wheat with the T. timopheevii cytoplasm. The same
authors indicated that the next less effective restorer gene is
located on the 4RL chromosome. Several genes controlling the
production of pollen in the CMS–T. timopheevii system have
been reported on six chromosomes of Triticum aestivum
L. (Rf1-1A; Rf2-7D; Rf3-1B; Rf4-6B; Rf5-6D; Rf7-7B) and
the effective Rf3 locus located on the 1BS chromosome has
been precisely mapped (Ma and Sorrells 1995; Kojima et al.
1997). Quantitative trait loci (QTL) analysis allowed for the
localisation of additional less efficient restorers on chromosomes 2A, 4B and 6A (Ahmed et al. 2001). In rye (the second
180
ancestor of the triticale), QTL analyses performed on F2 mapping populations resulted in the detection of genes restoring
male fertility in CMS systems on chromosomes 1R, 3R, 4R,
5R and 6R (Miedaner et al. 2000; Stojałowski et al. 2004).
Different molecular markers, like RAPD, ISSR, SSR,
AFLP and SCAR, were applied for genetic research on
triticale (Masojć 2000; Stojałowski and Góral 2002; Góral
et al. 2005; Tams et al. 2005; Stojałowski et al. 2006) but
none of these techniques resulted in the construction of a
genetic map. The development of Diversity Arrays Technology (DArT, Jaccoud et al. 2001) opened up new possibilities
in the exploration of triticale genome, because it allowed for
the construction of the first genetic maps covering the entire
genome of this artificial species (Tyrka et al. 2011; Alheit et
al. 2011). This study was aimed at the application of DArT
markers for mapping the triticale genome, as well as the
utilisation of a developed linkage map for the localisation
of genes restoring male fertility in plants carrying the T.
timopheevii cytoplasm.
The F2 population used in this study consisted of 414
individuals and was developed by the pollination of male
sterile inbred line CMS-Salvo 15/1 by restorer line Stan I.
The male sterile maternal component of the cross was kindly
provided by H. Góral (The Agricultural University in Krakow,
Poland) and the pollinator line was obtained from A.
Łukaszewski (University of California, Riverside, CA, USA).
All individuals of F1 progeny were fully male fertile. Seeds of
the F2 population were harvested after the self-fertilisation of
one F1 plant.
Individual plants of the F2 progeny were grown with a
25×25-cm spacing during the 2010/11 vegetation season in
a field belonging to the West Pomeranian University of
Technology in Szczecin, Poland. In two subsequent days
with stable weather conditions, the evaluation of male fertility was conducted via visual scoring of two to three spikes
per plant at the flowering stage, according to the 5-step scale
proposed by Góral et al. (2006), where 1 corresponds to
absolute sterility and 5 to full fertility of anthers. In addition,
three to five spikes of each plant were bagged before
flowering and visual observations of fertility were verified
after harvest, by the evaluation of seed setting under isolation.
DNA was isolated from young leaves harvested in early
spring and kept in a deep freezer at −70 °C. The GenElute
Plant Mini Kit from Sigma-Aldrich was employed for this
purpose. Marker analyses were performed on parental lines,
as well as on 92 individuals taken from the segregating
F2 population. The group of segregants consisted of plants
representative of two opposite phenotypes: male sterile
(MS) and male fertile (MF). As only 12 fully sterile individuals were identified in the whole population, the MS
group was supplemented by 26 partially male sterile plants
that produced few (1–19) grains per spike. The MF group
consisted of 54 completely fertile individuals (producing not
J Appl Genetics (2013) 54:179–184
less than 54 grains per spike and visually scored as 5 on the
valuation scale). Only plants revealing a consistent assessment by visual scoring and observation of seed setting under
bags were considered for the marker analyses. These were
performed with the use of DArT (Jaccaud et al. 2001).
The genetic map of triticale was constructed with the use
of JoinMap® 3.0 software (Van Ooijen and Voorrips 2001).
The localisation of DArT markers on triticale and rye genetic
maps reported by Alheit et al. (2011), Milczarski et al. (2011)
and Tyrka et al. (2011) were applied for the assignment of
linkage groups to entire chromosomes. For grouping analyses,
the LOD=4.0 level was initially established, but in some
cases, when markers formerly mapped on different chromosomes were included in the same linkage group, the LOD
level was increased to 5.0 or 6.0.
Agreement of the distribution of male sterility within the
studied population (data obtained by visual scoring and
analysis of seed setting under bags) with the normal population was tested using the Lilliefors test. Interval mapping,
the permutation test and the Kruskal–Wallis test (K-W test)
were performed with the use of MapQTL® 5.0 software,
revealing relationships between segregations of molecular
markers and male fertility restoration (Van Ooijen 2004).
Putative localisation of the gene(s) controlling male fertility
restoration was considered when the LOD value exceeded
3.0, and additional thresholds of significance for declaring
the presence of a given QTL were estimated from 1,000
permutations of the data (Doerge and Churchill 1996). As
suggested by the author of the software (Van Ooijen 2004),
during analyses using the K-W test, molecular markers were
considered to be significantly associated with the studied
trait if P<0.01.
Within the studied F2 population containing over 400
individuals, high phenotypic variation was observed. The
number of kernels produced within bagged spikes ranged
from 0 to 103. Twelve plants were fully male sterile. Partially male sterile plants were also not numerous, leading to
a limited number of MS genotypes available for marker
analyses. About 20 % of the population consisted of fully
fertile individuals, but the most numerous group contained
plants classified as partially male fertile (over half of the
entire studied population). The results of visual assessment
and analyses of seed setting in isolated spikes were consistent in the majority of cases, but some contradictories were
also noticed (such individuals were not considered for mapping analyses). Generally, the correlation coefficient between
results obtained by two applied methods of sterility/fertility
estimation was 0.55. The distribution of male sterility/fertility
(independent of the method of assessment—visual vs. seed
setting on bagged ears) deviated significantly from the normal
distribution, but these deviations were relatively small in the
case of seed setting data (data not shown). For this reason,
the results of seed setting under bags were chosen for the
J Appl Genetics (2013) 54:179–184
performance of an interval mapping analysis of loci responsible for the restoration of male fertility.
DArT resulted in 822 polymorphic markers. The segregation of 57 DArT markers deviated significantly from the
expected 3:1 ratio, but due to the selection of individuals for
mapping from groups of opposite phenotypes, these markers
were not excluded from the linkage analyses. Grouping analysis allowed for the construction of 24 linkage maps (Fig. 1).
The number of markers within particular linkage groups varied widely: chromosome 1A was represented by only four
linked markers but 99 loci of DArT markers were located on
the 6R chromosome. No group assigned to the 4A chromosome was obtained. Due to the homeology of chromosomes
within the triticale genome, some linkage maps could not be
clearly associated with a given chromosome. The coverage of
rye chromosomes by DArT markers was significantly more
efficient than chromosomes belonging to the wheat A and B
genomes. In total, the constructed genetic map of the triticale
genome contains 759 DArT markers and cumulatively spans a
distance of 974.4 cM (Fig. 1).
Interval mapping analysis revealed six genomic regions
associated with the determination of male fertility of triticale
181
with T. timopheevii cytoplasm (Table 1, Fig. 1). The most
effective genes were identified on chromosomes 6A, 6B and
6R. Their estimated additive effects ranged from approximately 20 to 30 grains per spike (differences in kernel
number produced within a bagged spike) and restorer alleles
originating from paternal component (line Stan I) of the
studied hybrid. All these QTL detected using the interval
mapping method were confirmed when the non-parametric
K-W test was applied (Table 1). The most efficient as
regards to the restoration of fertility seems to be a gene
located on the 6R chromosome-the only gene that was
statistically significant when the permutation test was
utilised. The next two loci detected by means of the interval
mapping method were located on chromosome 1B and the
linkage group ambiguously assigned to chromosomes 3A or
3B. Both genes revealed an additive effect at approximately
10 kernels per self-pollinated ear, but alleles increasing male
fertility seemed to be introduced by the maternal line CMSSalvo 15/1. Localisations of these genes were not confirmed
by the K-W test. The less effective QTL was detected by the
interval mapping method (but not by the K-W test) on the
3A linkage group. Calculated variance explained by all of
Fig. 1 Linkage groups of Diversity Arrays Technology (DArT) markers and localisation of genomic regions determining male fertility restoration
in the F2 generation of the cross [CMS-Salvo 15/1×Stan I] of winter triticale with the Triticum timopheevii cytoplasm
182
J Appl Genetics (2013) 54:179–184
Fig. 1 (continued)
the identified QTL exceeded 20 %, but these values are
probably overestimated for two reasons: the lack of codominant markers on the linkage maps and the selection
of opposite phenotypes for map construction and interval
mapping analyses. Some additional markers not indicated
by the interval mapping method were considered as being
significantly associated with male fertility by the K-W test.
These markers were located on chromosomes 5B, 1R and
4R (Fig. 1).
The restoration of male fertility in triticale with T.
timopheevii cytoplasm remains under the control of several
Rf genes (Góral et al. 2010), but information about their
Table 1 Characteristics of quantitative trait loci (QTL) controlling the restoration of fertility in the [CMS-Salvo 15/1×Stan I] F2 intercross (results
based on the seed setting data)
Chromosome
Interval mapping
Kruskal–Wallis test
Interval (cM)a
Maximum value
of LOD (position
in cM)b
Additive effect
of maternal
allele
Variance
explained
(%)
Kmaxc
Marker revealing
the max. value
of the K statistic
Mean value of
the genotype
CMS-Salvo
15/1
Mean value
of genotype
Stan I
3A
3A/3B
6A
1B
6B
7.91–11.86
1.00–4.89
14.8–59.2
0.00–5.30
11.7–44.7
13.08 (7.91 cM)
3.97 (3.89 cM)
6.03 (44.2 cM)
6.72 (1.5 cM)
4.64 (27.0 cM)
1.59
13.84
−23.51
10.77
−20.09
93.3
77.3
28.8
79.8
25.6
1.10NS
0.50NS
18.86**
5.08NS
11.85*
XwPt0398
XwPt8855
XwPt7445
XwPt3172
XwPt1241
33.5
42.5
16.5
28.6
22.5
44.3
37.4
50.3
45.3
52.8
6R
11.4–64.8
21.08 (29.2 cM)
−30.00
93.6
33.18**
XtPt3774
15.5
61.5
a
Mapping interval where LOD>3.0
b
Entry in bold indicates significance in the permutation test
c
Kmax maximum value of the K statistic within the interval; NS not significant; *Significant at P<0.001; **Significant at P<0.0001
J Appl Genetics (2013) 54:179–184
characterisation and localisation is still very limited. The
present study attempted to broaden knowledge concerning
this problem. It should be noted, however, that the above
results were obtained on the basis of the phenotyping of
individual plants in only one environment. The significance
of genotype by environment interactions for the detection of
QTL controlling different traits is usually investigated by the
assessment of “immortal” mapping populations (recombinant
inbred lines, DH lines) or by the verification of results from F2
in the F3 generation. Unfortunately, research on male sterility
restoration is frequently limited to the one generation
(Miedaner et al. 2000; Stojałowski et al. 2004) because the
reproduction of male sterile genotypes by self-pollination is
impossible. Application of the partial F3 generation obtained
from only fertile plants can be recommended for the identification of heterozygous F2 plants, which is reasonable for
investigations on one effective restorer gene (Börner et al.
1998; Hackauf et al. 2012). The assessment of male fertility
in different localisations but in the same year of study is
sometimes performed by vegetative cloning of individual
plants (Miedaner et al. 2000; Hackauf et al. 2012). The sensitivity of pollen production to variable environmental conditions (Góral et al. 2006) leads to the recommendation that the
male sterility/fertility of triticale plants carrying a sterilising
cytoplasm should be assessed in different environments. On
the other hand, it was stated also that donors of the most
effective non-restoring alleles are environmentally insensitive
genotypes (Góral et al. 2006). Geiger et al. (1995) reported
that the influence of environment on male sterility/fertility in
rye is the most significant for partially fertile individuals. This
rule probably also applies to triticale. Therefore, with the aim
to increase the proportion of variation affected only by plant
genotype, we decided to ignore all intermediates and to analyse, via molecular markers, only extreme (male sterile and
male fertile) phenotypes selected within the studied mapping
population. Additionally, Carey and Williamson (1991) as
well as Xu et al. (2005) indicated that the selection of individuals representing opposite phenotypes from the whole
F2 population should significantly increase the powerful
detection of QTL controlling the studied trait.
Obviously, the results of the present study should be
considered as preliminary; however, their close coincidence
with previous reports investigating the localisation of restorer
genes in T. timopheevii cytoplasm is intriguing. To the present
day, the only research focusing on the localisation of rye
genome genes acting as restorers in T. timopheevii cytoplasm
indicated the importance of 6RL and 4RL chromosomes
(Curtis and Lukaszewski 1993). In wheat with the same type
of sterilising cytoplasm, chromosomes 1BS, 2A, 4B and 6A
were reported as containing Rf loci (Ma and Sorrells 1995;
Kojima et al. 1997; Ahmed et al. 2001). The results of our
study on the cross [CMS-Salvo 15/1×Stan I] confirm that the
6R chromosome is the most significant as regards to male
183
sterility restoration in triticale with the T. timopheevii cytoplasm. Within the studied population, the remaining chromosomes of the sixth homeologic group (6A and 6B) were also
found to be important for male fertility. The lowest effect on
pollen fertility was identified for loci located on the 1B and 3A
(optionally 3B) chromosomes. The short arm of chromosome
1B might carry a restorer gene Rf3, but this effective allele
introduced into wheat from Triticum spelta (Kojima et al.
1997; Ahmed et al. 2001) was not detected among the hybrid
F2 generation investigated in the present study.
The presented research is of preliminary character and its
data need to be verified in further studies on a broader plant
material (e.g. vegetative clones of F2 plants or DH lines
carrying normal cytoplasm analysed by test-crosses with
CMS sources) examined under different environmental habitats. Nevertheless, the initial results obtained appear to confirm a complex genetic control of male fertility restoration in
triticale with T. timopheevii cytoplasm previously reported by
Góral et al. (2010). It is noticeable also that genes located on
all chromosomes of the sixth homeologic group are important
for the abundance of pollen production and the 6R chromosome is especially significant for this phenomenon.
Open Access This article is distributed under the terms of the Creative
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reproduction in any medium, provided the original author(s) and the
source are credited.
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